RNA replication is the central step in the infection cycles of plus-strand RNA viruses, which cause many diseases in humans, animals and plants. In spite of its significance, the mechanism of viral RNA replication is incompletely understood. Tractable model systems, such as tombusviruses, can contribute greatly to our understanding of viral RNA replication. Since the RNA-dependent RNA polymerase (RdRp), the key enzyme in replication, is similar in tombusviruses and numerous other important human and animal viruses (hepatitis C virus, flavivirus, including West Nile virus and dengue virus, pestiviruses) and plant viruses (luteo- and carmoviruses), understanding the mechanism of RNA replication in tombusviruses is expected to help studies on important, but less amenable RNA viruses of humans, animals and plants. In order to advance understanding of the mechanism of virus replication, the investigator has developed an efficient in vitro RNA replication assay based on purified tombusvirus RdRp from yeast. In addition, the investigator has established a novel RNA replicon based in vivo replication system in yeast. These advances make tombusviruses exceptionally suitable for studying the mechanism of RNA replication. One of the two essential replicase proteins coded by tombusviruses, termed p33, is an RNA chaperone protein, which influences many processes during infection, including replication of genomic RNA and Dl RNA, subgenomic RNA synthesis, RNA recombination and Dl RNA formation. The aims of this application is to determine the functions of p33 in tombusvirus replication, such as: (i) targeting the viral RNA template and the RdRp to the site of replication; (ii) template selectivity; (iii) stabilization of tombusvirus RNAs in cells; (iv) enhancement of synthesis of plus- and minus-strand RNAs; and (v) RNA structure unwinding and strand separation. The investigator will perform biochemical, cellular experiments using yeast and whole plants in order to define the multiple functions of p33 during tombusvirus infection. Overall, studies on tombusvirus replication will likely advance major scientific areas, including: (i) mechanism of RNA replication and interference; (ii) template and RdRp recruitment to the site of replication; (iii) regulation of RNA synthesis by essential protein factors; (iv) RNA-protein and protein-protein interactions during replication; (v) template selection by the viral replicase; and (vi) development of novel antiviral agents.